Apparatus for processing a fluid sample

ABSTRACT

This invention relates to an apparatus for an apparatus for processing a fluid sample comprising: (i) a sample processing chamber comprising a fluid inlet and a fluid outlet; (ii) a waste chamber downstream from the sample processing chamber and in fluid communication with the sample processing chamber fluid outlet and wherein the fluid communication between the sample processing chamber outlet and the waste chamber comprises a divergent analyte flow path; (iii) at least two further chambers up stream from the sample processing chamber both of which are in fluid communication with the sample processing chamber fluid inlet; (iv) a means for moving fluid from each of the at least two further chambers through the sample processing chamber and into the waste chamber or into the divergent analyte flow path as desired by applying positive or negative pressure to the desired flow path; and (v) a passive means for restricting the flow of fluid. This invention also relates to a method and use of the same.

This invention relates to an apparatus and associated method forprocessing a fluid sample.

The analysis of fluid samples, for example clinical or environmentalsamples, may be conducted for several reasons. One current area ofinterest is the development of a method to positively identifybiological material in a fluid sample, for example a clinical orenvironmental sample. This is important since it would help in the earlydiagnosis of disease states, which in turn would enable rapid treatmentand infection control, or the identification of environmentalcontaminants and the like. Although nucleic acid amplification, forexample the polymerase chain reaction (PCR) is a very useful andcommonly used method for positive identification of biological material,several problems exist when trying to successfully develop it for use ona day to day basis for the rapid identification of biological materialin many individual fluid samples in a non-laboratory environment, forexample to achieve near patient or point of care disease diagnosis.

One of the key problems lies in the fact that, prior to subjecting atypical clinical or environmental sample to nucleic acid amplification,it needs to undergo a sequence of processing steps using reagents, someof which are hazardous, to purify and concentrate the biologicalmaterial. However, nucleic acid amplification is just one of manydifferent possible examples of a technique where manipulation of asample, especially a fluid sample, is required which involves a numberof simultaneous or sequential processing steps. The processing stepsthemselves may be many and varied and may include for example chemical,optical, electrical, thermal, mechanical, acoustical, processing,sensing or monitoring, in addition to possible dilution andconcentration steps. To date such complex processing is conducted inlaboratories where samples are either treated manually one by one, or byusing specialist robotics facilities to process many different samplesin parallel. However, there are several problems associated with thesemethods. These include that they are slow, resource intensive,expensive, subject to error and to cross sample contamination. Inaddition, conventional fluid processing systems require fluid samples toflow sequentially through a series of different chambers where eachchamber is utilised for a single step in a sequence which may result inloss of sample, and automation of such processes requires the use ofcomplex fluidic assemblies and processing algorithms.

As such there remains a need to develop an improved apparatus whereby afluid sample can be processed using a series of pre-determinedsequential steps, to obtain a desired end product. Such an apparatusshould be readily adapted for use in a non-laboratory environment and byan operator with little or no laboratory training such that it can beused to manipulate a fluid sample, for example a clinical orenvironmental sample, prior to analysis, for example by nucleic acidamplification. Such an apparatus would ensure that analytical resultscould be rapidly obtained, would free the skilled worker from repetitivetasks and would reduce costs. Furthermore such an apparatus should havesufficient consistency and accuracy to prevent the failure of latertests, should be cheap to produce, and disposable, to minimise thelikelihood of cross contamination and to eliminate the need to steriliselarge amounts of equipment.

A prior art search has identified U.S. Pat. No. 6,374,684 whichdiscloses a fluid control and processing system comprising a pluralityof chambers and a moveable valve body that can be used to facilitate theprocessing of a fluid sample according to a given protocol. Althoughthis provides a development in the field of an apparatus for processinga fluid sample, several problems remain. One such problem is that, inorder to expose the fluid sample sequentially to different solutions, itis necessary to rotate the valve body to connect in turn, via severalexternal ports, a sample processing chamber with a reservoir of eachsolution. Such an apparatus is not well suited for use in anon-laboratory environment by a non-skilled laboratory worker because,for among other reasons, there is a need to connect the external portsto reservoirs of each required solution which is impractical and in thecase of hazardous chemicals may be a safety risk. In addition such anapparatus, due to its complexity, has a high associated cost, and isunlikely to be cost effective as a disposable apparatus and that resultin the potential for cross sample contamination. Furthermore, theapparatus utilises a single fluid displacement chamber to deliver eachprocessing solution to the sample in turn, and to remove any wastematerials, which may result in mixing of residual material in the fluiddisplacement chamber and potential failure of sensitive processingsequences. There remains a need to develop an apparatus for processing afluid sample that overcomes the above problems.

WO 00/62931 discloses a microfluidic apparatus with a sample inlet portconnected via a microchannel to a detection module. Fluidic movementwithin the apparatus is controlled by on or off chip pumps which applyan electric field. The apparatus can optionally comprise a storagemodule, a waste module, reaction modules and the like and theapplication discloses as an example the use of the apparatus for anucleic acid amplification reaction. Such an apparatus is useful forconducting nucleic acid amplification reactions on micro-scale volumesamples but the problem remains as to how to develop such an apparatuswhich can be used in the field wherein the volume of the sample isseveral millilitres as is the case for clinical or environmentalsamples.

Furthermore U.S. Pat. No. 6,391,541 discloses a cartridge for separatinga desired analyte from a fluid sample comprising a sample flow path, asample lysing chamber, a waste chamber, an analyte flow path and a flowcontroller for directing fluid flow. Again the patent discloses as anexample the use of the apparatus for the separation of a nucleic acidsample from a fluid sample. Again this apparatus presents a developmentin the field of processing a fluid sample but several problems remain.These include that in order that the sample may be processed in thedesired manner, including the desired chemical and physical processingsteps, the apparatus comprises complex fluidic channels. This hasrequired the use of many valves through the apparatus which requiremechanical opening/closing in order to ensure that the fluid proceeds inthe desired manner. Furthermore this complex arrangement may result insome sample remaining inside the fluid cartridge thereby reducing theamount of sample that is ultimately processed. This can lead toinaccurate results in the case of high volumes of sample which retain alow concentration of the desired analyte. Finally the complexity of thecartridge makes the apparatus more difficult to operate in the field bythe unskilled user.

An apparatus, and associated method, have now been developed whichovercome the above problems. The apparatus comprises a sample processingchamber comprising a fluid inlet and a fluid outlet; a waste chamberdownstream from the sample processing chamber and in fluid communicationwith the sample processing chamber fluid outlet and wherein the fluidcommunication between the sample processing chamber outlet and the wastechamber comprises a divergent analyte flow path; at least two furtherchambers up stream from the sample processing chamber both of which arein fluid communication with the sample processing chamber fluid inlet; ameans for moving fluid from each of the at least two further chambersthrough the sample processing chamber and into the waste chamber or intothe divergent analyte flow path as desired by applying positive ornegative pressure to the desired flow path; and a passive means forrestricting the flow of fluid.

A fluid sample is introduced into the sample processing chamber,optionally via a sample chamber where it may optionally interact with afunctional agent. One or more fluid processing solutions are then movedconcomitantly or sequentially from at least one of the further chambers,through the sample processing chamber where they interact with the fluidsample, and then into a waste chamber. By ensuring that the furtherchambers are both in communication with the sample processing chamber,the fluids can move through the apparatus without the need for eachfluid communication route to be established in turn. By controlling themeans for moving fluid through the fluid communication routes it ispossible to ensure that each fluid passes via a pre-determined protocolinto the sample processing chamber and if necessary, it is possible tominimise the interaction of each fluid with another prior to reachingthe sample processing chamber. By utilising a means for the applicationof positive or negative pressure to move fluid through the apparatus thecorrect fluid flow paths can be established with minimum complexity andby using a passive means for restricting the flow of fluid the fluidpathways can be simply controlled. The apparatus can be enhanced inseveral ways. These include optionally utilising a combination ofapplication of positive pressure and negative pressure, ie a vacuum, tomove fluids through the apparatus in a controlled manner; pre-loadingchambers with any reagents or fluids during production thus eliminatingthe need for the user of the apparatus to handle such materials; usingmore than one passive valve to prevent the back flow of fluids throughthe apparatus; and integrating a collection chamber into the apparatussuch that the processed sample can be collected directly for furtheruse, for example for nucleic acid amplification. In addition, one ormore of the chambers may optionally be adapted such that one or more ofthe fluids, including the fluid sample, may also be subjected tophysical processing for example thermal, acoustic, optical, electricalprocessing, sensing or monitoring techniques.

The apparatus of the present invention has several advantages. Theseinclude that it can be easily designed to accommodate a wide variety ofpre-determined processing sequences, including chemical and physicalsteps, to provide an easy to use fluid processing device; the apparatusutilises a single processing chamber thus minimising sample loss; theapparatus, including all chemical reagents and any waste produced, iscompletely integrated in a single unit therefore minimising samplecontamination and reducing exposure of the user to potentially hazardousmaterials; due to the use of a reduced number of moving parts theapparatus is likely to have increased reliability and simplicity of useeven for a user with little or no laboratory training; and it is cheapto manufacture which means that the apparatus can be designed to bedisposable further reducing the risk of sample cross contamination.

It is an object of the present invention to develop an apparatus, andassociated method, for processing a fluid sample. It is a further objectof this invention to design such an apparatus which is capable ofsubjecting a fluid sample to a series of sequential chemical or physicalprocessing steps in a pre-determined sequence, preferably to purify andconcentrate a fluid sample prior to a nucleic acid amplificationreaction. It is another object of this invention to design such anapparatus to be simple to use by a worker with little or no laboratorytraining in a non-laboratory environment. It is yet another object ofthis invention to design the apparatus such that any required chemicalsor waste product remain integrated within the apparatus to minimise userexposure to the sample, chemicals or waste. It is yet another object ofthis invention to design such an apparatus to be cheap to manufacturesuch that it can be disposed of after a single use thus reducing thelikelihood of sample cross contamination and eliminating the need forsterilising large amounts of equipment. These, and other objects of thisinvention, will become apparent in light of the following disclosure.

SUMMARY OF THE INVENTION

According to a first aspect this invention relates to an apparatus forprocessing a fluid sample comprising:

-   -   (i) a sample processing chamber comprising a fluid inlet and a        fluid outlet;    -   (ii) a waste chamber downstream from the sample processing        chamber and in fluid communication with the sample processing        chamber fluid outlet and wherein the fluid communication between        the sample processing chamber outlet and the waste chamber        comprises a divergent analyte flow path;    -   (iii) at least two further chambers up stream from the sample        processing chamber both of which are in fluid communication with        the sample processing chamber fluid inlet;    -   (iv) a means for moving fluid from each of the at least two        further chambers through the sample processing chamber and into        the waste chamber or into the divergent analyte flow path as        desired by applying positive or negative pressure to the desired        flow path; and    -   (v) a passive means for restricting the flow of fluid.

According to a second aspect this invention relates to a method ofprocessing a fluid sample comprising:

-   -   (i) placing the sample in the sample processing chamber of an        apparatus according to the present invention;    -   (ii) applying a positive or negative pressure to move fluid        through the apparatus;    -   (iii) subjecting the sample to one or more processing steps; and    -   (iv) collecting the processed sample from the divergent analyte        flow path.

According to a third aspect this invention relates to the use of anapparatus according to the present invention for purification andconcentration of nucleic acid material from a fluid sample.

DETAILED DESCRIPTION OF THE INVENTION

All publications cited herein are hereby incorporated by reference intheir entirety, unless otherwise indicated.

As used herein the term “fluid communication” means that a path throughwhich fluid can in principle flow exists between the chambers inquestion although fluid flow may be restricted by one or more passivemeans for restricting the flow of fluid.

As used herein the term “continuous fluid communication” means that apath through which fluid can flow exists at all times between thechambers in question.

As used herein the term “a passive means for restricting the flow offluid” shall be taken to mean a means that restricts fluid flowingthrough a given path but wherein the restriction can be overcomepassively namely purely by the application of positive or negativepressure to either the means or to the flow path itself, therebyallowing fluid flow along the given path. Optionally such means may beopened by passive action or closed by passive action. Furthermore suchmeans may allow flow of fluid in either direction through the fluid flowpath in which the means is situated depending on the position ofapplication of a pressure or alternatively the means may only allow theflow of fluid in a single direction through the fluid flow path.

As used herein the term “fluid sample” means any sample that exists as agas, a liquid, a solution comprising a sample solvated by a solvent, ora fluid system comprising one or more phases for example an emulsion. A“fluid sample” is also taken to mean a sample which may initially beintroduced into the apparatus as a solid or a viscous liquid but whichis then diluted or dissolved by the adding of a volume of solvent.

As used herein the term “functional agent” means a solid chemical orphysical agent which is used in the apparatus or method of the presentinvention. It may comprise one or more chemical reagents dosed as asolid powder, bead, capsule, pressed tablet and the like comprising areagent to interact with the fluid sample. Suitable examples of suchreagents include, but are not limited to, lysis reagents for examplechaotrophic salts, nucleic acid targets, nucleic acid syntheticcontrols, bacteriophage, lyophilised enzymes, dyes, detergents and thelike. However, the term functional agent should also be understood tocomprise physical means for interacting with the fluid sample. Thesecould include, but are not limited to, a magnetic stirrer bead, aheating means, magnetic beads coated with antibodies and the like.

The elements of the apparatus are described in more detail below.

This invention relates to an apparatus for processing a fluid samplecomprising a sample processing chamber, a waste chamber, at least twofurther chambers, a means for moving fluid from each of the at least twofurther chambers through the sample processing chamber and into thewaste chamber or divergent analyte flow path; and is characterised inthat each of the at least two further chambers are in fluidcommunication with the sample processing chamber and in that the sampleprocessing chamber is in fluid communication with the waste chamber.Embodiments of the invention facilitate processing of a fluid sampleaccording to a pre-determined protocol.

The sample processing chamber is the region of the apparatus in whichthe fluid sample itself is subjected to one or more processing steps.These processing steps can include chemical processing steps such asdiluting the sample, washing the sample sequentially with one or morebuffer solutions, reacting the sample with one or more chemicalreagents, but may also include physical steps for example radiating thefluid sample with one or more thermal radiation, subjecting the fluidsample to acoustical processing and the like. The sample processingchamber may optionally comprise a an active member, preferably atrapping member selected from the group consisting of a microfluidicchip, a solid phase material, a filter, a filter stack, an affinitymatrix, a magnetic separation matrix, a size exclusion column, acapillary tube and mixtures thereof. It is preferred that the sampleprocessing chamber comprises a trapping member capable of trapping thebiological material in a fluid sample for example capable of trappingcells, spores, viruses, large or small molecules, proteins and the like.The exact trapping member chosen will depend on the nature of thematerial to be trapped. The preferred trapping member for use herein isa glass fibre filter that extends across part or all of the internalsurface area of the sample processing chamber.

The sample processing chamber comprises a fluid inlet and a fluid outletand is in fluid communication with at least two further chambers on theone hand and a waste chamber on the other hand. The waste chamber isdown stream from the sample processing chamber and in fluidcommunication with the sample processing chamber fluid outlet and the atleast two further chambers are up stream from the sample processingchamber both of which are in fluid communication with the sampleprocessing chamber fluid inlet. At least one of these fluidcommunications may be a continuous fluid communication. The fluidcommunication between the sample processing chamber outlet and the wastechamber comprises a divergent analyte flow path. Again this may be acontinuous fluid communication. The apparatus also comprises a means formoving fluid from the at least two further chambers through the sampleprocessing chamber into the waste chamber or into the divergent analyteflow path as desired by applying positive or negative pressure to thedesired flow path. It is preferred that the means for moving fluid fromeach of the two further chambers through the sample processing chamberand into the waste chamber or the divergent analyte flow path moves thefluid sequentially from the first at least two further chambers throughthe sample processing chamber and into the waste chamber and then fromanother further chambers through the sample processing chamber and intothe waste chamber or the divergent analyte flow path.

Many different means are acceptable for moving the fluid. The meansmoves fluid by the application of either a positive pressure or anegative pressure to the desired flow path. One example of a means forthe application of negative pressure is a means for generating a vacuumthat is attached to the apparatus via an outlet port, for example anoutlet port on the waste chamber or an outlet port on the divergentanalyte flow path and that is able to draw fluid through the apparatusas desired. The vacuum can be attached to the outlet port using one ofseveral different means known to one of ordinary skill in the art. Oneeffective way of attaching the vacuum to the outlet port is by the useof bellows since these can be readily attached to even a small outletport giving a high tolerance and reducing the need for very accuratehandling. Such an adaptation is particularly useful if the apparatus isto be operated mechanically or by a non-skilled worker. It is preferredthat the waste chamber of the apparatus comprises an outlet port towhich a means for generating a vacuum can be attached. This has theresult that the vacuum is able to draw the fluid from at least one ofthe further chambers, through the sample processing chamber and into thewaste chamber. It is also preferred that the divergent analyte flow pathcomprises an outlet port such that a means for generating a vacuum canbe readily attached here. The means for generating a vacuum can also beadapted in several ways. For example it can be adapted to draw the fluidthrough the apparatus in a pre-determined sequence. Optionally theapparatus can be designed to comprise a passive means for restrictingthe flow of fluid through the apparatus such that an increased vacuumforce is required to sequentially release the fluids from each chamber,such that by increasing the vacuum during use it is possible tosequentially draw the fluids from first one chamber and then another forexample by controlling the dimensions of fluid flow paths or byincorporating membranes or gates which need to be passively opened toallow fluid flow. The use of a vacuum can also be adapted to pull airthrough the apparatus as an interim step between the moving of eachfluid such that the fluid communication routes are cleared and thedifferent fluids do not interact with each other prior to entering thesample processing chamber. This can be important in sample processingprotocols that are very sensitive for example where the interaction of afirst buffer with a second prior to entering the sample processingchamber may neutralise its effects, or where it is important that thewhole of a very small volume of material reaches the sample. If a vacuumis used it is also preferred to consider that any material within theapparatus may be aerosolised by the vacuum and drawn out of theapparatus into the atmosphere. If the apparatus is adapted for use withreagents which may prove a safety hazard if released it is important toadapt the apparatus to minimise or eliminate release of the aerosolisedmaterial. One such possible adaptation is to incorporate filtermembranes into the apparatus, or into the means for applying a vacuum,such that the aerosolised material is captured and not released into theatmosphere.

An example of another suitable means for moving fluid through theapparatus is a means for applying positive pressure or force behind thefluid. Again such a force can be applied by many means known to one ofordinary skill in the art. One example is the use of a plunger in one ofmore of the further chambers, the depression of which would expel anyfluid in that a chamber through the fluid communication routes into thesample processing chamber and then into the waste chamber. Again, theuse of a means for applying force behind the fluid, for example by theuse of a plunger, can be adapted such that the fluids from the at leasttwo further chambers move concomitantly or sequentially, preferablysequentially, from the further chamber through the sample processingchamber and into the waste chamber or the divergent analyte flow path.For example it would be possible to sequentially depress one or more ofa series of plungers to move fluid sequentially from a first furtherchamber through the sample processing chamber into the waste chamber andthen from a another and so on. In both instances described it ispossible that the means for moving the fluid could be provided manuallyby the use of syringes, plungers, pumps and the like or mechanically byintegrating the apparatus of the present invention into a furtherapparatus able to provide the power and means as required. Again such ameans for applying positive pressure can be used in conjunction with apassive means for restricting the flow of fluid through the apparatus.

It is preferred in the apparatus of the present invention to utilise acombination of both a means for generating a vacuum and a means forapplying force behind the fluid to move fluid from at least one of thefurther chambers through the sample processing chamber and into thewaste chamber. The use of the two together has the advantage that theinitial force behind the fluid initiates release of the fluid from anygiven chamber that the vacuum could direct the fluid flow through theapparatus thus preventing it from being diverted from the desired path.This enables the apparatus to be readily designed such that fluid canflow sequentially from the further chambers through the sampleprocessing chamber according to a pre-determined protocol.

The fluid communication routes of the present invention are provided byone or more channels that pass through the apparatus connecting thechambers in the desired manner. The apparatus is designed such that atleast two further chambers are in fluid through the sample processingchamber and into the waste chamber or the divergent analyte flow path.For example it would be possible to sequentially depress one or more ofa series of plungers to move fluid sequentially from a first furtherchamber through the sample processing chamber into the waste chamber andthen from a another and so on. In both instances described it ispossible that the means for moving the fluid could be provided manuallyby the use of syringes, plungers, pumps and the like or mechanically byintegrating the apparatus of the present invention into a furtherapparatus able to provide the power and means as required. Again such ameans for applying positive pressure can be used in conjunction with apassive means for restricting the flow of fluid through the apparatus.

It is preferred in the apparatus of the present invention to utilise acombination of both a means for generating a vacuum and a means forapplying force behind the fluid to move fluid from at least one of thefurther chambers through the sample processing chamber and into thewaste chamber. The use of the two together has the advantage that theinitial force behind the fluid initiates release of the fluid from anygiven chamber that the vacuum could direct the fluid flow through theapparatus thus preventing it from being diverted from the desired path.This enables the apparatus to be readily designed such that fluid canflow sequentially from the further chambers through the sampleprocessing chamber according to a pre-determined protocol.

The fluid communication routes of the present invention are provided byone or more channels that pass through the apparatus connecting thechambers in the desired manner. The apparatus is designed such that atleast two further chambers are in fluid communication with the sampleprocessing chamber. These chambers are optionally designed to each havean individual outflow channels which connect at a common point prior toentering the sample processing chamber. It is preferred that there isonly a single entry point for solutions to pass into the sampleprocessing chamber, ie the sample processing chamber inlet, to allow forreduced design complexity. As such it is preferred that the outflowchannels from each further chamber meet and then flow into a commonsample processing channel prior to entering the sample processingchamber. One or more of these fluid communications may be a continuousfluid communication.

In order to minimise the flow of fluid through the chamber in aninappropriate manner it is preferred that the apparatus comprises apassive means for restricting the flow of fluid through the apparatuswhich is designed to contain features which limit any particular fluidflow route until such time as that flow route is required. Failure to dothis could result in fluid flowing from more than one chambersimultaneously and thus could destroy the sample processing sequence orcould result in fluid flowing from the waste chamber back through theapparatus. Examples of suitable means to control such flow include useof one or more of membranes covering channels that are broken whenpressure is applied behind them or in front of them, very small diameterchannels through which a fluid is unable to flow due to its surfacetension without the application of a force, pre-filling any chamberscomprising buffer solutions using suction which then acts to hold such afluid in place until a force is applied to release it, using valvesthroughout the apparatus which are operated by vacuum, pressure, magnetsand the like, designing the fluid communication routes to comprise asmall reservoir which needs to be filled completely in order for fluidcommunication to be established which allows for small leaks to beaccommodated without overflows, and the like. It is preferred that theapparatus comprise one or more of such features to ensure appropriatefluid flow. In the apparatus of the present invention it is preferredthat the passive means for restricting the flow of fluid comprises areservoir located in the fluid communication between at least one of theat least two further chambers and the sample processing chamber. It isalso preferred that the passive means for restricting the flow of fluidcomprises a fluid pathway of small diameter such that fluid can not flowthrough the pathway without the application of a positive or negativepressure, located in the fluid communication between at least one of theat least two further chambers and the sample processing chamber. It iseven more preferred that the continuous fluid communication between atleast one of the at least two further chambers comprises both of thesefeatures. Such examples of passive means for restricting the flow offluid allow continuous fluid communication pathways to be establishedwithin the apparatus whilst at the same time controlling fluid flowwithin the apparatus. This further reduces the complexity of theapparatus.

The sample processing chamber is similarly in fluid communication with awaste chamber via a waste channel. It is also important that material inthe waste chamber is not able to flow back into the sample chamber. Itis therefore preferred to design the apparatus to incorporate a passivemeans for restricting the flow of fluid between the sample processingchamber and the waste chamber. It is preferred that this comprises apassive valve, preferably a 1-way valve, located in the fluidcommunication between the sample processing chamber and the wastechamber to prevent such back flow. It is preferred that the passivevalve is down stream of the divergent analyte flow path. One simplesolution is to utilise a small bead, for example a glass bead, that ispositioned in the opening of the waste channel at a point in the fluidpath connecting it to the sample processing chamber. When the apparatusis in use and fluid is moving from the sample processing chamber to thewaste chamber the movement of fluid, or the use of a vacuum, willrelease the bead from the opening thus allowing fluid flow. When thereis no fluid flow the bead will return to sit in the opening of the wastechannel thus preventing the back flow of liquid through the apparatus.It is preferred that the application of a vacuum via the waste chamberis used to operate this valve.

Another example of a passive means for restricting the flow of fluid isto control the flow of fluid by the use of gravity. It is preferred thatprior to entering the sample processing chamber the fluid is directed,preferably by a vacuum, up through a pre-processing channel. The fluidthen enters the sample processing chamber from above where it can fallby gravity through the chamber. Again it is preferred that when thefluid leaves the sample processing chamber via the waste channel itagain passes up and enters the waste chamber from the top. Thisarrangement can also prevent fluid flowing backwards through theapparatus eg from the waste chamber into the sample processing chamberand from the sample processing chamber into the further chambers.Depending on the particular use in question, one of ordinary skill inthe art would be able to design the apparatus to comprise any necessaryfluid flow control mechanisms selected from those mentioned and others.

The waste material from the sample processing is collected in a wastechamber. It is preferred that the waste chamber is fully integrated intothe apparatus of the present invention such that additional bottles arenot required for collecting and then disposing of such waste. This isespecially preferred if the materials in question are either hazardousor infectious since this minimises the need for user handling. The wastechamber should have sufficient volume to be able to readily hold all ofthe fluids used during the sample processing. It is preferred that thewaste chamber is housed within any redundant space inside the apparatusbetween and around the sample processing chamber and the at least twofurther chambers. This enables the most efficient use of space thuskeeping the overall size of the apparatus to a minimum. As mentionedpreviously it is preferred that the waste chamber comprises an outletwhich can be connected to a means for generating a vacuum such that avacuum can be applied to the apparatus to direct fluid flow directlythrough the apparatus into the waste chamber.

The apparatus also comprises at least two further chambers. Depending onthe use of the apparatus these further chambers may have several roles.Possible examples of such chambers may include a buffer chamber whichcomprises a buffer solution or water which are required in the sampleprocessing protocol or a sample chamber into which a sample, either as afluid or a solid, may be initially introduced into the apparatus andwhich may optionally comprise a first reagent with which the sampleinteracts, or optionally where a solid sample is initially dissolved ina solvent, or alternatively where the sample may be subjected tophysical processing. The chambers can be pre-filled with the requiredsolutions or reagents during manufacture. This has several advantagesincluding that the user need not be concerned with accurately measuringaliquots of chemical solution from bulk, the chambers do not themselvesneed to be attached to a reservoir of solution and the chambers can bepre-loaded filled with an air pocket which, when the fluid is drawnthrough the apparatus, can follow the fluid flow to ensure that thefluid channels are cleared prior to the use of a subsequent fluid. Thechambers may optionally comprise internal partial barriers, for examplea plastic spindle extending through part of the internal chamber whichcan be used to minimise the movement of solid reagents within thechamber if a vacuum or force is applied. Similarly the chambers maycomprise a membrane to prevent early release of the contents ordisruption, for example by contamination or evaporation, of the contentsduring prior sample processing steps. In one embodiment of the presentinvention it is preferred that at least one of the at least two furtherchambers is pre-filled with a buffer solution selected from the groupconsisting of an aqueous solution of potassium acetate andTris.hydrochloride, or an aqueous ethanolic solution of potassiumacetate and Tris.hydrochloride. It is also preferred that at least oneof the at least two further chambers acts as a sample chamber whichcomprises an inlet port though which a sample can be introduced into theapparatus. Furthermore it is preferred that the sample chamber comprisesa reagent, preferably a reagent comprising a lysis reagent, morepreferably chaotrophic salts. This may be in the form of a solid bead orfreeze dried onto one or more surfaces inside the chamber.

The chambers of the apparatus, including one or more of the furtherchambers and or the sample processing chamber itself, can be designed ifrequired such that the contents of the chamber can be subjected tophysical steps in the processing sequence. For example the walls of thechamber may comprise heating elements which allow their contents to bewarmed, they may be flexible to allow acoustic processing, they may betransparent to one or more wavelengths of light to allow opticalprocessing, sensing or monitoring and the like. It is preferred that atleast one of the chambers of the apparatus is coated with anelectrically conducting polymer such as that disclosed in WO98/24548. Ifsuch physical processing is required the apparatus should be designedsuch that the chamber is positioned for easy and efficient access to thesource of the physical processing. For example the chamber may bepositioned towards the external face of the apparatus or may extendpartially or fully outside of the main body of the apparatus such that apart of the chamber is able to interact with a source of the physicalprocessing eg a light source or a heating source. It is preferred thatat least one chamber of the apparatus is located externally to the mainbody of the apparatus whilst remaining in fluid communication with thesample processing chamber and it is further preferred that this chamberhas walls which are coated at least partially with an electricallyconducting polymer. Examples of chambers which may be preferentially belocated externally to the main body of the apparatus include any chamberin which the analyte or any buffers are required to be heated.

In addition the apparatus may also be designed to comprise one or morefilter membranes. As already discussed it is preferred that the sampleprocessing comprise a trapping member. Again, as already discussed, ifthe apparatus comprises an outlet which is attached to a means forgenerating a vacuum it is preferred that a filter is incorporated eitherin the apparatus or in the means for generating a vacuum to preventaerosolised material being released into the atmosphere. Furthermore theapparatus may comprise other filters for example in the communicationroutes to prevent solid particulate causing blockages. Another optionaluse of a filter could be at the inlet port of the sample chamber tofilter a sample prior to entering the apparatus. Alternatively, if theapparatus comprises an inlet port through which air is drawn from theatmosphere into the apparatus it may be necessary to use a filtermembrane to ensure that any contamination from the atmosphere does notenter the apparatus and potentially contaminate the sample beingprocessed. It is preferred that the sample chamber inlet port comprisesa filter membrane which can be positioned either before or after thesample has been introduced into the sample.

The apparatus of the present invention may optionally comprise acollection chamber into which the processed sample can be directlycollected. The chamber may be integrated into the apparatus or theapparatus may be designed such that the chamber can be simply andsecurely clipped into place when required. When in place such a chamberwould be downstream of the analyte flow path and in fluid communicationwith the divergent analyte flow path outlet and thereby the sampleprocessing chamber. The apparatus could be operated such that the samplecould be directed into this chamber using a means for moving fluidwithin the apparatus. For example the apparatus would also be providedwith a means for moving the processed sample from the sample processingchamber into the collection chamber. It would therefore be possible todirect fluid from the sample processing chamber into the collectionchamber by disconnecting the means for moving fluid into the wastechamber and connecting the means for moving fluid into the collectionchamber. It is preferred that such a means comprise a means forgenerating a vacuum connected to the collection chamber via a secondoutlet, in this instance where the path of the vacuum flows from thesample processing chamber through the collection chamber thus divertingany fluid from the waste chamber but instead into the collectionchamber. As mentioned previously, if a vacuum is used it may beadvantageous to fit a filter membrane into the apparatus upstream of thevacuum to prevent aerosolised material from the sample entering theatmosphere. As with other chambers the collection chamber can bedesigned such that it can be subjected to physical processing, sensingor monitoring and may extend either in whole or part outside of the mainbody of the apparatus in order to facilitate the interaction of thecollection chamber with a source of the physical processing. It ishighly preferred that the collection chamber is adapted for use in anucleic acid amplification. It is therefore preferred that thecollection chamber is external to the main body of the apparatus,comprises walls which are at least partially coated with an electricallyconducting polymer to facilitate thermal cycling of the sample and alsocomprises a transparent section through which the nucleic acidamplification reaction can be optically monitored, preferably byfluorescence. Alternatively the collection chamber may be detachablesuch that, once the processed sample is collected, it can be removed foruse elsewhere. One of the main advantages of an integrated, ifdetachable, collection chamber, is that the processed sample can becollected without the need for any intervention or additional apparatus.Again this simplifies the apparatus for the user, minimises cross samplecontamination and minimises user exposure to the sample. However if thecollection chamber is to be pre-dosed with reagents which degrade it maybe useful to store the collection chamber separately and attach it tothe apparatus when required. Examples of such reagents include thoseknown by one skilled in the art to be required for a nucleic acidamplification reaction and detection system for example nucleic acidprimers, nucleic acid probes, fluorescing dyes, enzyme buffers,nucleotides, magnesium salts, bovine serum albium, denaturants, and thelike.

In some circumstances it may be necessary that, once the main sampleprocessing protocol is complete, that the sample further interacts withyet another reagent prior to being collected in the collection chamber.This final step could be provided by optionally including in theapparatus a further chamber, a post processing chamber between thesample processing chamber and the collection chamber. After theprocessed sample has been eluted from the sample processing, it entersthe post processing chamber and interacts with a reagent such as thatrequired for a reverse transcriptase step in reverse transcriptasepolymerase chain reaction nucleic acid amplification. Optionally theapparatus could be provided with a post processing chamber downstreamfrom the collection chamber containing the final reagent in question. Itis then possible to apply a vacuum to draw the processed sample from thesample processing chamber through the collection chamber and into thepost processing chamber, allowing the processed sample to furtherinteract with any reagent therein and then disconnecting the vacuum andreapplying the vacuum through the waste chamber to draw the fullyprocessed sample back into the collection chamber. As with the otherchambers it is preferred that the apparatus is pre-dosed with any suchreagents to minimise the need for the user to have to handle thesematerials. When the apparatus is used for preparing a sample for nucleicacid amplification preferably the reagent comprises one or more reagentsselected from the group consisting of nucleic acid primers, nucleic acidprobes, fluorescing dyes, enzyme buffers, nucleotides, magnesium salts,bovine serum albium, denaturants, and the like.

The apparatus itself can have a wide variety of different designs,shapes, sizes and can be made of many different materials depending onthe specific use. In order to minimise the cost of the apparatus and toensure that it is economically feasible to produce for a single use itis preferred that the apparatus is manufactured from a cheap materialsuch as a thermoplastic material for example polyethylene orpolypropylene, polycarbonate, acrylic, nylon or butadiene-styrenecopolymer or mixtures thereof. It is preferred that the apparatus ismanufactured from as few components as possible. As such it is preferredthat the main bulk of the apparatus is manufactured as a singleinjection moulded unit containing the chambers and key channels. It ispreferred that the intricate channels are formed by the ultrasonicsealing of the base of the injection moulded unit to a plate containingthe channel routes. Any plungers are added later, as can be a lid, toprevent the leaking of any materials and the escape of contaminatedwaste after use. It is further preferred that the apparatus ismanufactured from a material which can be incinerated such that afteruse the apparatus, including any waste, can be easily disposed ofwithout the build up of waste or any risk of exposure of the user to thechemicals involved. The apparatus can be transparent or translucent.Advantageously any plungers can also be colour coded to help direct theunskilled user as to the correct use of the apparatus.

The apparatus may also be optionally designed such that it can integratewith further additional apparatus for example a sample bottle such thatthe fluid sample, once collected from the patient or environment, can beintroduced into the fluid processing apparatus without any spillage, ora sample collection cone such that the sample can be collected directlyinto the fluid processing apparatus. The two apparatus could integratevia the use of a seal for example a quick fit seal, a screw seal orother means. If the apparatus of the present invention is moulded fromplastic then such sealing devices can be integrated easily into theshape. This integration further simplifies the use of the apparatus in anon-laboratory environment for staff with little or no scientifictraining and again minimises user interaction with the sample itself.

Additionally the apparatus may integrate with a mechanical apparatus.This could be for several reasons including for applying the means formoving the fluid using a physical apparatus, such as the vacuum orplunger depression, or for subjecting one or more of the chambers of theapparatus to a physical processing step for example thermal, optical oracoustical processing, sensing or monitoring. If such integration isrequired it is important to ensure that the apparatus is designed suchthat it can integrate effectively with such an additional physicalapparatus. It is also important to ensure that such an integration is assimple as possible such that it can effectively be used by a non-skilledworker. This may include designing the apparatus such that it can onlybe integrated in a single orientation, using colour coding to aid theorientation and the like.

This invention also relates to a method of processing a fluid samplecomprising:

-   -   (i) placing the sample in the sample processing chamber of an        apparatus according to the present invention;    -   (ii) applying a positive or negative pressure to move fluid        through the apparatus;    -   (iii) subjecting the sample to one or more processing steps; and    -   (iv) collecting the processed sample from the divergent analyte        flow path.

The processing steps can be chemical steps or physical steps.

According to a third aspect this invention relates to the use of anapparatus according to the present invention for purification andconcentration of nucleic acid material from a fluid sample. Such asample should preferably be prepared such that it can then undergo anucleic acid amplification, for example polymerase chain reactionamplification.

FIGURES

This invention will now be described by reference to a specificembodiment of the apparatus of the present invention shown in thefollowing figures in which;

FIG. 1 shows a perspective view of the apparatus from the side;

FIG. 2 shows a perspective view of the apparatus from the base;

FIG. 3 shows a perspective view of the apparatus from the top, with thetop plate removed such that the internal chambers can be seen;

FIG. 4 shows an internal view of the base plate of the apparatus;

FIG. 5 is a cross sectional view of the apparatus along A-A showing thefirst step of use of the apparatus for processing a fluid sample forPCR;

FIG. 6 is a cross sectional view of the apparatus along B-B showing thesecond step of use of the apparatus for processing a fluid sample forPCR;

FIG. 7 is a cross sectional view of the apparatus along C-C showing thethird step of use of the apparatus for processing a fluid sample forPCR;

FIG. 8 is a cross sectional view of the apparatus along D-D showing thefourth step of use of the apparatus for processing a fluid sample forPCR; and

FIG. 9 is a cross sectional view of the apparatus along E-E showing thefifth step of use of the apparatus for processing a fluid sample forPCR.

FIG. 1 shows an apparatus of the present invention 2 comprising a samplechamber 4, a first buffer chamber 6 with a first plunger 8; a secondbuffer chamber 10 with a second plunger 12. The first plunger 8 and thesecond plunger 12 comprise sockets 34 that allow them to integrate withan external apparatus to move the plungers during use. The apparatus 2additionally comprises a small volume water chamber 14 that extendsbeyond the main body of the apparatus 2. This comprises a membrane sealenclosing the chamber such that the water within the chamber does notevaporate during storage or the initial stages of sample processing. Themembrane is a thin plastic sheet for example 0.1 mm-0.2 mm, skin ofacrylonitrile butadiene styrene. The apparatus also comprises a firstoutlet port 16 and a second outlet port 18 by which one or more vacuumscan be attached to the apparatus 2. The sample chamber 4 comprises a lid20 which is attached to the main body of the apparatus 2 via an arm 22which is able to pivot around a peg 24. After the introduction of thesample (not shown) into the sample chamber 4 the lid 20 is pivotedaround the peg 24 into position to seal the sample chamber 4. The lid 20also comprises an inlet outlet port 26 through which air can be drawninto the apparatus 2 via a pump (not shown). The apparatus 2 alsocomprises a channel 28 that extends from the bottom to the top of theapparatus 2 and which is external to the main body of the apparatus 2.This channel 28 forms part of the fluid communication network of theapparatus. The base plate 30 of the apparatus 2 and the top plate 32 ofthe apparatus 2 are manufactured as separate units and are fitted to theapparatus 2 during the final stages of manufacture.

Referring to FIG. 2, the apparatus 2 comprises a top plate 32, a baseplate 30, and an exterior channel 28. The view shows that the apparatushas three reservoirs 50, 52 and 54 that are also exterior to the mainbody of the apparatus 2. The reservoir 50 is below the sample chamber,the reservoir 52 is below the first buffer chamber and the reservoir 54is below the second buffer chamber. These reservoirs (50, 52 & 54) areused as part of the mechanism to prevent sample or buffer from each ofthese chambers flowing through the fluid communication network of theapparatus in an inappropriate manner. The base plate 30 of the apparatus2 also comprises a collection chamber 56 that is also exterior to themain body of the apparatus 2. Once the fluid sample (not shown) has beenfully purified it enters the divergent analyte flow path and then thecollection chamber 56 where it is able to undergo the PCR amplificationreaction. It is preferred that the collection chamber 56 is coated withan electrically conducting polymer (not shown) and that it comprises twotransparent faces 58 through which a nucleic acid amplification reactioncan be monitored.

Referring to FIG. 3, the apparatus 2 comprises a sample chamber 4, afirst buffer chamber 6 and a second buffer chamber 10. The apparatus 2comprises a waste chamber 100 that is the dead space within the mainbody of the apparatus surrounding the other internal chambers. Theapparatus comprises a sample processing chamber 102. This is in fluidcommunication with the sample chamber 4 and the buffer chambers 6, 10via a pre-processing channel 104. The sample processing chamber 102 isin fluid communication with the waste chamber 100 via a waste channel 28which links with the waste chamber 100 via the waste port 106. Theapparatus also comprises a post processing chamber 108 which is linkedto the second vacuum outlet port (18, not shown in this perspective) andalso to the sample processing chamber 102 (communication not shown).

Referring to FIG. 4, the base plate 30 comprises the sample reservoir50, a first buffer reservoir 52 and a second buffer reservoir 54. Italso comprises a sample channel 152, a first buffer channel 154 and asecond buffer channel 156, which provides the fluid communication fromthe sample chamber, the first buffer chamber, and the second bufferchamber (not shown) respectively to the sample processing chamber (notshown) via the pre-processing channel the base of which is shown at 158.The base of the sample processing chamber 160 is in fluid communicationwith the base of the waste chamber 162. The base of the sampleprocessing chamber 160 is also in fluid communication with the base ofthe post processing chamber 164 via the divergent analyte flow path andthe collection chamber (not shown).

Referring to FIG. 5, the sample 200 is introduced into the apparatus 2via the sample chamber 4. Once inside the sample chamber the samplechamber lid 20 is closed. This lid comprises a filter 202 that preventscontamination from the air entering the apparatus and also preventsaerosolised sample leaving the apparatus. In the sample chamber theapparatus interacts with a first reagent bead 204, which comprises lysisreagent containing chaotrophic salts, for example guanidiumhydrochloride, to lyse any bacteria within the sample. The first reagentmay optionally comprise a nucleic acid target that is later able to actas a means for normalising the efficiency of the subsequent PCRamplification reaction. After the sample has interacted with the firstreagent a vacuum is applied to the apparatus via the first vacuum outletport (vacuum shown in this cross section, but first vacuum outlet port16 is not shown). This vacuum draws the sample firstly into the samplechamber reservoir 50, along the sample channel 152 to the base of thepre-processing channel 158. The sample is then drawn up thepre-processing channel 104 and into the sample processing chamber 102.The sample processing chamber 102 comprises a filter means 206, forexample a glass fibre filter, that is capable of isolating from thesample any nucleic acid as the sample passes through. The vacuum thendraws the sample out of the filter to the base of the sample processingchamber 160, to the base of the waste channel 162 and then up into thewaste channel 28. At the top of the waste channel the sample, from whichall of the nucleic acid has been removed, enters into the waste chamber100 through the waste channel outlet 106. This figure does not show thewaste material in the waste chamber. The waste channel comprises a smallbead 208 that is positioned in the base of the waste channel 162. Whenthe vacuum is applied the bead 208 rises within the waste channel 28allowing the passage of fluid through the channel 28 into the wastechamber 100. When the vacuum is removed the bead 208 falls and coversthe base of the waste channel 162 thus sealing the entrance. Thisprevents waste fluid from flowing back into the sample processingchamber 102. This view of the apparatus also shows the water chamber 14,the first buffer chamber 6 and plunger 8 and the post processing chamber108, and the post processing chamber outlet port 18, but the fluidcommunication between these chambers are not shown in this view.

Referring to FIG. 6, the first buffer chamber 6 of the apparatus 2comprises the first buffer 252, for example an aqueous potassiumacetate/Tris.hydrochloride solution such as that available in PromegaWizard™ kit. A vacuum is applied to the apparatus via the waste chamberoutlet port (not shown but as in FIG. 5). Simultaneously the plunger 8is depressed within the first buffer chamber 6 which initiates the flowof the first buffer 252 through the reservoir 52 and into the sampleprocessing chamber 102 via the first buffer channel 154 and thepre-processing channel 104 which are linked at the base of thepre-processing channel 158. The first buffer channel 154 is designed tohave a very narrow diameter at the point it leaves the first bufferreservoir 52. This ensures that, due to the surface tension of the firstbuffer, it is unable to leak into the first buffer channel 154 untildepression of the plunger 8. Once in the sample processing chamber 102,as with the sample, the buffer flows through the filter means 206 thuswashing the nucleic acid material on the filter membrane. The firstbuffer 252 is then drawn by the vacuum (not shown) via the waste channel28, and the waste channel outlet port 106, into the waste chamber 100(waste material in the waste channel is not shown).

Referring to FIG. 7, the second buffer chamber 10 comprises the secondbuffer 302, for example an aqueous ethanolic solution of potassiumacetate/Tris.hydrochloride such as that available in Promega Wizard™kit. A vacuum is applied to the apparatus via the waste chamber outletport (not shown but as in FIG. 5). Simultaneously the plunger 12 isdepressed within the second buffer chamber 10 which initiates the flowof the second buffer 302 through the reservoir 54 and into the sampleprocessing chamber 102 via the second buffer channel 156 and thepre-processing channel 104 which are linked at the base of thepreprocessing channel 158. As before the second buffer channel 156 isdesigned to have a very narrow diameter at the point it leaves thesecond buffer reservoir 54. This ensures that the second buffer 302 doesnot leak. Again, once in the sample processing chamber 102, the bufferflows through the filter means 206 again washing the nucleic acidmaterial on the filter membrane. The second buffer 302 is then drawn bythe vacuum (not shown) via the waste channel 28, and the waste channeloutlet port 106, into the waste chamber 100 (waste material in the wastechamber is not shown).

Referring to FIG. 8, the apparatus is configured to air dry the filtermembrane comprising the purified nucleic acids to remove any excesssolvent from the filter. In order to do this a vacuum is applied to theapparatus 2 via the first vacuum outlet port 16 (not shown). This drawsair into the apparatus through the inlet port 26 in the sample chamberlid 20. This air passes through a filter 202 to remove material in theair thus preventing sample contamination. It is then drawn by the vacuumthrough the sample chamber 4, the sample chamber reservoir 50, thesample chamber channel 152, the pre-processing channel 104, the sampleprocessing chamber 102, the filter means 206, the waste channel 28 andout of the apparatus through the waste chamber 100. The means forapplying a vacuum to the apparatus 2 via the outlet port 16 is thenremoved.

Referring to FIG. 9, the small volume chamber 14 comprises purifiedwater, preferably approximately 10 μl. This is warmed using an externalmeans for heating (not shown) until the temperature of the water ispreferably greater than 80° C. A second vacuum is then applied to theapparatus via the second vacuum outlet port 18. A plunger 305 isdepressed releasing the warmed water from the water chamber 14 into thesample processing chamber 102. A cone 315 is used to direct the smallvolume of water directly to the filter means 206. The water is drawnthrough the filter means 206, eluting the purified nucleic acid materialas it passes through, into the collection chamber 56 by the secondvacuum. The second vacuum further draws the water through the divergentanalyte flow path, the collection chamber 56 and into the postprocessing chamber 108. The post processing chamber 108 contains asecond solid reagent 310 comprising further PCR reagents for exampleprobes, fluorescing dyes and further nucleic acid controls. This reagent310 dissolves in the solution. Removing the second vacuum then allowsthe solution to fall by gravity into the collection chamber 56.Alternatively the first vacuum can be reapplied to draw the solutionback into the collection chamber 56.

Once in the collection chamber the nucleic acids are ready to besubjected to the heat cycling required to conduct the PCR amplification.The collection chamber is preferably designed such that its walls aremade of a heat conducting polymer. The collection chamber can theneither be subjected to the heat cycling in situ or removed and placed ina further apparatus for cycling.

This embodiment of the apparatus has been developed to purify thenucleic acid material from a 10 ml fluid sample. The main body of theapparatus has a height of from about 70 to about 80 mm and has adiameter of from about 70 to about 80 mm. The sample chamber has avolume of about 20 ml and the first and second buffer chamber have avolume of about 30 ml. The small volume water chamber has a volume ofabout 500 μl and the sample processing chamber has a volume of about 2ml and has a diameter of about 5 mm. The pre-processing channel and thewaste channel each have a diameter of about 3 mm. The waste chamberwhich is formed by the dead space within the main body has a volume ofabout 120 ml. The fluid communication channels are formed byultrasonically welding a shaped base plate to the underside of thechamber. The channels have a diameter of about 1 mm. At the point wherethe channels leave the sample reservoirs this diameter is reduced toapproximately 0.6 mm to prevent unwanted fluid flow from the chambersinto the sample processing chamber.

1. An apparatus for processing a fluid sample comprising: (i) a sampleprocessing chamber comprising a fluid inlet and a fluid outlet; (ii) awaste chamber downstream from the sample processing chamber and in fluidcommunication with the sample processing chamber fluid outlet andwherein the fluid communication between the sample processing chamberoutlet and the waste chamber comprises a divergent analyte flow path;(iii) at least two further chambers up stream from the sample processingchamber both of which are in fluid communication with the sampleprocessing chamber fluid inlet; (iv) a means for moving fluid from eachof the at least two further chambers through the sample processingchamber and into the waste chamber or into the divergent analyte flowpath as desired by applying positive or negative pressure to the desiredflow path; and (v) a passive means for restricting the flow of fluid. 2.An apparatus according to claim 1 wherein the means for moving fluidfrom at least one of the at least two further chambers through thesample processing chamber comprises a means for generating a vacuum. 3.An apparatus according to claim 2 wherein the waste chamber comprises anoutlet port which is connected to the means for generating a vacuum. 4.An apparatus according to claim 2 wherein the analyte flow pathcomprises an outlet port which is connected to the means for generatinga vacuum.
 5. An apparatus according to claim 1 wherein the means formoving fluid from at least one of the at least two further chambersthrough the sample processing chamber comprises a plunger capable ofbeing depressed to expel fluid from the at least one further chamber. 6.An apparatus according to claim 5 wherein the means for moving fluidfrom at least one of the two further chambers through the sampleprocessing chamber additionally comprises a means for generating avacuum.
 7. An apparatus according to claim 1 wherein the means formoving fluid from each of the two further chambers through the sampleprocessing chamber moves the fluid sequentially from the first at leasttwo further chambers through the sample processing chamber and into thewaste chamber and then from the second at least two further chambersthrough the sample processing chamber and into either the waste chamberor the divergent analyte flow path.
 8. An apparatus according to claim 1wherein the passive means for restricting the flow of fluid comprises avalve located in the fluid communication between the sample processingchamber and the waste chamber.
 9. An apparatus according to claim 8wherein the valve is down stream of the divergent analyte flow path. 10.An apparatus according to claim 8 wherein the valve comprises a beadwhich is opened by applying a positive or negative pressure.
 11. Anapparatus according to claim 1 wherein the passive means for restrictingthe flow of fluid comprises a reservoir located in the fluidcommunication between at least one of the at least two further chambersand the sample processing chamber.
 12. An apparatus according to claim 1wherein the passive means for restricting the flow of fluid comprises afluid pathway of small diameter such that fluid can not flow through thepathway without the application of a positive or negative pressure,located in the fluid communication between at least one of the at leasttwo further chambers and the sample processing chamber.
 13. An apparatusaccording to claim 1 comprising a collection chamber downstream of theanalyte flow path and in fluid communication with the analyte flow pathoutlet.
 14. An apparatus according to claim 13 wherein the collectionchamber comprises a reagent, preferably a reagent comprising one or morenucleic acid amplification reagents, more preferably a reagent selectedfrom the group consisting of nucleic acid primers, nucleic acid probes,fluorescing dyes, enzyme buffers, nucleotides, magnesium slats, bovineserum albumen, and denaturants.
 15. An apparatus according to claim 13wherein collection chamber comprises an outlet port which optionally maybe connected to a means for generating a vacuum.
 16. An apparatusaccording to claim 15 wherein the apparatus comprises a post processingchamber down stream from the collection chamber in fluid communicationwith the collection chamber outlet and which optionally itself comprisesan outlet which may be connected to a means for generating a vacuum. 17.An apparatus according to claim 1 wherein the sample processing chambercomprises an active member, preferably a trapping member selected fromthe group consisting of a microfluidic chip, a solid phase material, afilter, a filter stack, an affinity matrix, a magnetic separationmatrix, a size exclusion column, a capillary tube, and mixtures thereof.18. An apparatus according to claim 17 wherein the sample processingchamber comprises a glass fibre filter membrane.
 19. An apparatusaccording to claim 1 wherein at least one of the at least two furtherchambers is pre-filled with a buffer solution, preferably a buffersolution selected from the group consisting of an aqueous solution ofpotassium acetate and Tris.hydrochloride, or an aqueous ethanolicsolution of potassium acetate and Tris.hydrochloride.
 20. An apparatusaccording to claim 1 wherein at least one of the at least two furtherchambers acts as a sample chamber comprising an inlet port through whicha sample is introduced into the apparatus.
 21. An apparatus according toclaim 20 wherein the sample chamber inlet port comprises a filtermembrane.
 22. An apparatus according to claim 20 wherein the samplechamber comprises a reagent, preferably a reagent comprising a lysisreagent, more preferably a chaotrophic salt.
 23. An apparatus accordingto claim 1 wherein the apparatus comprises at least one chamber locatedexternally to the main body of the apparatus.
 24. An apparatus accordingto claim 23 wherein the chamber located externally to the main body ofthe apparatus is the collection chamber.
 25. An apparatus according toclaim 23 wherein the chamber located externally to the main body of theapparatus is at least one of the at least two further chambers.
 26. Anapparatus according to claim 23 wherein at least one chamber locatedexternally has walls which are coated with an electrically conductingpolymer.
 27. A method of processing a fluid sample comprising: (i)placing the sample in the sample processing chamber of an apparatusaccording to claim 1; (ii) applying a positive or negative pressure tomove fluid through the apparatus; (iii) subjecting the sample to one ormore processing steps; and (iv) collecting the processed sample from thedivergent analyte flow path.
 28. Use of an apparatus according to claim1 for purification and concentration of nucleic acid material from afluid sample.
 29. Use according to claim 28 wherein the nucleic acidmaterial is then subjected to a polymerase chain reaction amplification.